Methods of screening agonistic antibodies

ABSTRACT

Cell strains with ligand-dependent (factor-dependent) proliferation are super-infected with antibody libraries against each of the various receptor chains. Antibody genes are recovered from strains that autonomously grow by the autocrine stimulation by agonistic antibodies formed by appropriate combinations, enabling effective screening of agonistic antibodies. In this method, effective screening can be carried out using antibodies as libraries. Furthermore, the manipulations are simple, and there is no need for complicated operations.

TECHNICAL FIELD

The present invention relates to novel methods of screening foragonistic antibodies.

BACKGROUND ART

Antibodies in the blood are highly stable, and since they have noantigenicity, they are drawing much attention as pharmaceuticals. Ofthese, it has been a while since bispecific antibodies, which cansimultaneously recognize two types of antigens, have been proposed, butonly those that merely bind two types of antigens exist at present.However, since antibodies bind to specific epitopes within antigens, itmay be possible to place two antigens at desirable distances and anglesby selecting appropriate antibody combinations.

In many cytokine receptors, it is thought that the angle and length ofchains that form homo/hetero-dimers change when a ligand binds, thusenabling the receptors to transmit signals into cells. Thus, appropriateanti-receptor antibodies can mimic receptor dimerization initiated byligand-binding, and become potential agonistic antibodies. Monoclonalantibodies that display agonistic activity against MPL, a homodimer,have already been reported (Blood 1998 Sep. 15; 92(6): 1981-8,US98/17364). However, to obtain such agonistic antibodies, selectionmust be made from a huge range of antibodies, requiring effectiveselection methods.

In conventional assays, it is necessary to select antibodies that bindto antigens, i.e., receptor chains, and add these antibodies to anappropriate cell assay system that responds to the ligands. This becomesparticularly troublesome where the receptors form heterodimers.Antibodies against each of the two chains (A, B) that form the receptormust be selected, and every combination of A and B must be tested one byone. In addition, to generate bivalent antibodies, it is necessary tofuse antibody-producing hybridomas, or to construct expression vectorsfor all of the antibodies, and introduce all combinations thereof intocells. Examination of 100 types of antibodies against each of the A andB chains necessitates the testing of 10,000 types of combinations, andrequires a total of 400 types of expression vectors for L and H chainsto be constructed and introduced into cells 10,000 times. There are alsomethods that use libraries that provide antibodies displayed on phagesas bispecific diabodies. However, since the direct addition of E. coliculture supernatant to cell culture systems has a bad effect on cells,purification becomes necessary and monospecific diabody contamination(theoretically 50%) becomes inevitable.

DISCLOSURE OF THE INVENTION

The present invention was achieved in view of the above. An objective ofthe present invention is to provide efficient novel methods of screeningfor agonistic antibodies, especially multi-specific agonisticantibodies. More specifically, it aims to provide methods of screeningfor agonistic antibodies that utilize autocrine cell growth.

With the above objective, the present inventors conducted extensiveexperiments. As a result, they discovered effective methods of screeningfor agonistic antibodies, such as the methods below. In these methods,cell strains that proliferate in a ligand-dependent manner are infectedwith anti-receptor antibody libraries. Antibody genes are recovered fromstrains that replicate autonomously upon autocrine stimulation due tothe binding of agonistic antibodies, and agonistic antibodies areprepared based on the recovered genes. When the antibodies aremulti-specific agonistic antibodies, cell strains with ligand-dependentgrowth are super-infected with antibody libraries against each of thevarious receptor chains. Antibody genes are recovered from strains thatreplicate autonomously upon autocrine stimulation due to the binding ofthe multi-specific agonistic antibodies in appropriate combinations, andmulti-specific agonistic antibodies are generated based on the recoveredgenes. More specifically, this can be carried out, for example, asfollows:

Mice are first immunized with either A-chain or B-chain receptors, andmRNAs are extracted from the splenocytes of these animals. L-chain andH-chain variable regions are recovered by RT-PCR using primers thatcorrespond to mouse CDRs. Single chain variable regions (scFvs) aresynthesized by assembly PCR to construct a phage library.Antigen-binding antibody clones are concentrated by panning, thesynthesized single chain variable regions obtained from concentratedclones are inserted between a signal sequence for animal cells andCH1-hinge-CH2-CH3 and, to construct a library that is integrated intoplasmids to be used for producing retroviruses. By expressing chimericchains comprised of a target receptor chain and the G-CSFR intracellulardomain, Ba/F3 cells, whose proliferation depends on the binding of theligand to target chimera receptor, are prepared. These cells areinfected with anti-A-chain antibody library viruses. These cells arefurther infected with anti-B-chain antibody library viruses, andcultured following the washing and removal of ligands (factors). Cellsthat now reproduce ligand (factor)-dependently are recovered and cloned,and agonistic activity is confirmed by using the culture supernatantsand physiological assay systems. The antibody CDR genes incorporated inthe chromosomes of the clones are recovered using PCR, and applied tothe production of multi-specific agonistic antibodies. In this method,effective screening can be carried out using antibodies as libraries.Furthermore, the manipulations are simple, and there is no need forcomplicated operations.

As described above, the present inventors developed novel methods thatcan effectively screen for agonistic antibodies, thus completing thepresent invention.

In other words, the present invention relates to novel methods that caneffectively screen for multi-specific agonistic antibodies. Morespecifically, the present invention provides:

[1] a method of screening for agonistic antibodies that comprises thefollowing steps (a) to (c):

(a) providing a cell that expresses a multimer-forming receptor and atest antibody, where the cell grow depending on the corresponding ligandof the receptor;

(b) determining the test antibody to comprise agonistic activity whenautocrine cell growth is autonomous; and

(c) selecting those antibodies that comprise agonistic activity;

[2] the method of [1] that further comprises the step of introducing agene that encodes the heavy chain of the test antibody into the cell ofstep (a) having been introduced with a gene that encodes the light chainof the test antibody and a gene that encodes the receptor;

[3] the method of [1] or [2] where the receptor is a chimeric receptorwith a protein that comprises a function of transducing a cell growthsignal;

[4] the method of any one of [1] to [3] where the receptor is adimer-forming receptor;

[5] the method of [4] where the dimer-forming receptor is a homo-dimer;

[6] the method of [4] where the dimer-forming receptor is ahetero-dimer;

[7] the method of any one of [1] to [6] where the protein that comprisesthe function of transducing a cell growth signal is a G-CSF receptor;

[8] the method of any one of [1] to [7] that comprises the introductionof an antibody library to the cell;

[9] the method of [8] where the antibody library is a retroviralantibody library;

[10] the method of any one of [1] to [9] where the test antibody is amulti-specific antibody;

[11] the method of [10] that comprises linking the test antibody's heavyand light chain variable regions with a linker;

[12] the method of [11] that comprises producing the antibody withvariable regions linked by a linker, using a method that comprises thesteps (a) to (c):

(a) producing a single chain Fv against the first receptor chain;

(b) producing a single chain antibody against the first receptor chainby linking the single chain Fv with a CH1-hinge-CH2-CH3; and

(c) producing a multi-specific antibody that comprises the single chainantibody produced in step (b);

[13] the method of [11] that comprises producing the antibody with itsvariable regions linked by a linker, using a method that comprises thesteps (a) to (c):

(a) producing a single chain Fab against the first receptor chain;

(b) producing a single chain antibody against the first receptor chainby linking the single chain Fab with an Fc; and

(c) producing a multi-specific antibody that comprises the single chainantibody produced in step (b);

[14] a method of screening for an agonist multi-specific antibody thatcomprises the steps (a) to (c):

(a) contacting between a multi-specific antibody and a receptorcomprising a first receptor chain and a second receptor chain, where themulti-specific antibody comprises a variable region that can bind withthe first receptor chain and a variable region that can bind with thesecond receptor chain;

(b) determining whether the test multi-specific antibody comprisesagonistic activity; and

(c) selecting antibodies that comprise agonistic activity;

[15] the method of [14] that comprises expressing the receptor and thetest multi-specific antibody in the same cell;

[16] the method of [15] where the cell is a cell that grows depending onthe corresponding ligand of the receptor;

[17] the method of [15] or [16] where the receptor comprises thefunction of transducing a cell growth signal;

[18] the method of [17] where the receptor is a chimeric receptor with aprotein that comprises the function of transducing a cell growth signal;

[19] the method of [18] where the protein that comprises the function oftransducing a cell growth signal is a G-CSF receptor;

[20] the method of any one of [15] to [19] where the test multi-specificantibody is determined to comprise agonistic activity when autocrinecell growth is autonomous;

[21] the method of any one of [15] to [20] that further comprises thestep of introducing an antibody library against the first receptor chainand the second receptor chain into the cell, respectively;

[22] the method of [21] where the antibody library is a retroviralantibody library;

[23] the method of any one of [14] to [22] that comprises linking thelight chain variable regions and heavy chain variable regions of themulti-specific antibody with a linker;

[24] the method of [23] that comprises producing a multi-specificantibody with variable regions linked by a linker, using a method thatcomprises steps (a) to (c):

(a) producing a single chain Fv against the first receptor chain;

(b) producing a single chain antibody against the first receptor chainby linking the single chain Fv with a CH1-hinge-CH2-CH3; and

(c) producing a multi-specific antibody that comprises the single chainantibody produced in step (b);

[25] the method of [23] that comprises producing the multi-specificantibody with variable regions linked by a linker, using a method thatcomprises steps (a) to (c):

(a) producing a single chain Fab against the first receptor chain;

(b) producing a single chain antibody against the first receptor chainby linking the single chain Fab with an Fc; and

(c) producing a multi-specific antibody that comprises the single chainantibody produced in step (b);

[26] the method of any one of [14] to [25] that comprises theintroduction of “Knobs-into-holes” by amino acid substitution at the CH3region of the multi-specific antibody;

[27] the method of any one of [14] to [26] where the multimer of thereceptor is a heterodimer;

[28] the method of any one of [14] to [27] where the multi-specificantibody is a bispecific antibody;

[29] a method for producing an agonistic antibody comprising steps (a)to (c):

(a) screening for an agonistic antibody by a method of any one of [1] to[28];

(b) introducing a gene that encodes the agonistic antibody selected bythe screening of step (a) into a host cell;

(c) recovering the agonistic antibody from the host cell of step (b) orits cell culture supernatant;

[30] a cell that expresses an antibody, and a receptor that multimerizesby binding with the antibody, where the cell grow depending on thecorresponding ligand of the receptor;

[31] the cell of [30] where the receptor is a chimeric receptor with aprotein that comprises the function of transducing a cell growth signal;

[32] the cell of [30] or [31] where the antibody is a multi-specificantibody;

[33] the cell of any one of [30] to [32] where the receptor that ismultimerized by binding with the antibody comprises the function oftransducing a cell growth signal;

[34] a multi-specific agonistic antibody that comprises the linking ofthe light chain variable region and heavy chain variable region bylinkers, and the introduction of “Knobs-into-holes” by amino acidsubstitution at the CH3 region of the antibody.

In the present invention, set terms have been defined to simplifyunderstanding of the terms used within the present specification. Itshould be understood that these definitions should not be used to limitthe present invention.

The present invention provides effective novel methods of screening foragonistic antibodies. In these methods, test antibodies comprisingvariable regions that can bind receptors are contacted with thosereceptors (step (a)).

In the present invention, when the antibodies are multi-specificantibodies, multi-specific test antibodies comprising variable regionsthat can bind the first and second receptor chains are contacted withthose receptors (step (a)).

“Agonistic antibody” refers to an antibody that comprises an agonisticactivity against a given receptor. In general, when an agonist ligand(factor) binds to a receptor, the tertiary structure of the receptorprotein changes, and the receptor is activated (when the receptor is amembrane protein, a cell growth signal or such is usually transducted).If the receptor is a dimer-forming type, an agonistic antibody candimerize the receptor at an appropriate distance and angle, thus actingsimilarly to a ligand. An appropriate anti-receptor antibody can mimicdimerization of receptors performed by ligands, and thus can become anagonistic antibody.

“Multi-specific antibodies” refer to antibodies that can bindspecifically with a number of types of antigens. That is, multi-specificantibodies are antibodies that comprise specificities to at least twodifferent types of antigens. Usually, such molecules bind with twoantigens (i.e., a bispecific antibody). However, in the presentspecification, “multi-specific antibody” also comprises antibodies thatcomprise specificities against more antigens (for example, three types).A multi-specific antibody can be a full-length antibody or a fragmentthereof (for example, F(ab′)₂ bispecific antibody). Multi-specificantibodies are useful in clinical fields such as immunodiagnostics,therapy, and diagnoses using immunological assays. Suitable examples ofthe multi-specific antibodies of the present invention includebispecific antibodies that can specifically bind to two types ofantigens. Usually, when the antigens are hetero-receptors, bispecificantibodies will recognize each of the two polypeptide chains that makeup the hetero-receptor.

In the present invention, “multi-specific agonistic antibodies” refer toantibodies that comprise a quality (characteristic) of both theabove-mentioned agonistic antibodies and multi-specific antibodies.

In a preferable embodiment of the present invention, the test antibodiesfor use in the screening methods of the present invention are firstprepared. However, the process for preparing the test antibodies is notessential. The test antibodies can be known compounds, or antibodymolecules (or antibody-like molecules) that exist in nature, orfragments thereof, and such.

In the present invention, there are no limitations as to what kinds ofantibodies can be used as test antibodies, but multi-specific antibodiesare preferable, and bispecific antibodies are even more preferable.

Equally, there are no particular limitations as to what kinds ofreceptors can be used as the receptors to which the agonistic antibodiesof the present invention bind and transmit signals. Preferable examplesof the receptors include cell membrane receptors, more preferablyreceptors that form multimers, and even more preferably receptors thatform dimers (e.g., heteroreceptors).

The above-mentioned test antibodies of the present invention can usuallybe prepared by immunizing animals with antigens. The antigens used toimmunize the animals include complete antigens that have immunogenicity,and incomplete antigens (including hapten) having no antigenicity. Inthe present invention, receptors for which agonistic antibodies to bescreened are thought to act as ligands are used as antigens (immunogens)mentioned above. There are no specific limitations as to the abovereceptors, but usually they are multimers, and preferably dimers. Dimersinclude homodimers, which are made of identical receptor chains, andheterodimers, which are made of different receptor chains. Heterodimersare more preferable. Examples of the immunized animals that can be usedinclude mice, hamsters, rhesus monkeys, and such. Immunization of theseanimals with the antigens can be carried out by methods known to thoseskilled in the art. In the present invention, the antibody L-chain andH-chain variable regions are preferably recovered from the immunizedanimals or cells of those animals. This process can be carried out usingtechniques generally known to those skilled in the art. The animalsimmunized by the antigens express antibodies against those antigens,particularly in splenocytes. Thus, for example, mRNA can be preparedfrom the splenocytes of immunized animals, and using primers thatcorrespond to the CDR of those animals, the L-chain and H-chain variableregions can be recovered by RT-PCR. Here, “CDR” refers to three regions(CDR1, CDR2 and CDR3) that directly and complementarily bind theantigen, and are present in the hyper-variable region of antibodyvariable regions. Examples of primers that can be used as primers thatcorrespond to the CDR include primers that correspond to a frameworkwith less variation than the CDR, or primers that correspond to thesignal sequences, and CH1 and CL. In addition, lymphocytes can also beimmunized in vitro. After this, DNAs that encode the antibodiescontained in the spleens of immunized animals, or in lymphocytes, can beisolated by conventional methods, for example, methods using nucleotideprobes and such that can bind specifically to the genes encodingantibody heavy and light chains.

The receptors that act as immunogens can be the entire proteins thatmake up those receptors, or peptide fragments of those proteins. In apreferable embodiment of the present invention, if the receptor is aheteroreceptor, two or more different types of peptide chain fragmentsare used as immunogens for the peptide chain fragment making up one partof the heteroreceptor. In the present invention, the different peptidechain fragments are called the “first receptor chain” and the “secondreceptor chain” respectively. Where a receptor of the present inventionforms a dimer, the above-mentioned “first receptor chain” and “secondreceptor chain” are preferably peptide chains (or fragment peptidethereof) that respectively make up each subunit of that dimer. Forexample, when a heterodimer is made up of two types of peptide chains,the A-chain and the B-chain, it is preferable that the A-chain (or apeptide fragment thereof) be the “first receptor chain”, and the B-chain(or a peptide fragment thereof) be the “second receptor chain”. Byimmunizing animals using these first and second receptor chains asimmunogens, test antibodies can be prepared that comprise variableregions which can bind those first and second receptor chains.

Depending on the situation, the immunogens used to immunize the animalscan also be soluble antigens that bind with other molecules, orfragments thereof. When using receptor-like transmembrane molecules asthe antigens, it is preferable to use their fragments (for example, theextracellular region of a receptor). The immunogens can also be cellsthat express transmembrane molecules on the cell surface. Such cells canbe naturally derived (tumor cell lines, etc.) or may be thoseconstructed to express a transmembrane molecule using recombinanttechnologies.

In a preferable embodiment of the above screening methods of the presentinvention, cells whose growth depends on ligands (factors) of thereceptors for which the agonistic antibodies act as agonists, are firstinfected with a viral antibody library against each of the receptors.Cells infected with that library produce anti-receptor antibodies. Inthe present invention, the antibodies produced by the above-mentionedcells are supplied as “test antibodies” for the screening methods of thepresent invention. When the antibodies are multi-specific antibodies,the cells whose growth depends on ligands (factors) of the receptors forwhich the agonistic antibodies act as agonists, are super-infected withviral antibody libraries against each of the different types of receptorchains. Cells infected with these libraries produce a variety ofanti-receptor antibodies that arise from appropriate peptide chaincombinations.

The above-mentioned cells of the present invention are usuallyeukaryote-derived cells, preferably animal cells, and more preferablyhuman-derived cells. In a preferable embodiment of the presentinvention, cells expressing test antibodies also express the abovereceptors (the receptors for which agonistic antibodies act asagonists). Thus, a preferable embodiment of the present inventioncomprises expressing a receptor and a test antibody in the same cell. Ifa test antibody secreted from a cell binds with that receptor andcomprises agonistic activity against a receptor, the receptor wouldtransduce a cell growth signal and consequently, the cell would undergoautonomous autocrine replication. “Autonomous autocrine replication”refers to the phenomenon whereby cells replicate independently using asubstance produced by the cell itself as a growth signal. Multi-specificagonistic antibodies can be screened using the presence or absence ofautonomous autocrine replication as an index. In a preferable embodimentof the present invention, when cells expressing a test antibody andreceptor undergo autonomous autocrine replication, the test antibody canbe determined to comprise agonistic activity.

Agonistic activity of the test antibodies of the present invention canbe determined using the indexes below:

(1) Whether or not a ligand (factor)-dependently growing cell will growin the same way when a test antibody is added during cell culture aswhen a ligand (factor) is added. If the cell grows, the test antibody isdetermined to comprise agonistic activity.

(2) Whether or not a cell line with intrinsic ligand (factor)-dependentactivities (not limited to growth) shows the same reaction when a testantibody is added during cell culture as when a ligand (factor) isadded. If the cell line shows the same reaction as for a ligand(factor), the test antibody is determined to comprise agonisticactivity.

In the present invention, cells transducing the above-mentioned cellgrowth signals usually express the receptors for which the antibodiesselected by the screening methods of the present invention act asagonists on the cell surface. These cells transduce cell growth signalsby binding with the ligands of those receptors (for example, agonisticantibodies). Thus, in the present invention, cells that are used arepreferably cells that can proliferate receptor ligand(factor)-dependently (cells with ligand (factor)-dependentproliferation). Preferably, on binding with a ligand, the receptors ofthe present invention usually transduce cell growth signals. However,when the receptors of the present invention are of a type that do nottransduce cell growth signals, they can be used in the present inventionas so-called “chimeric receptors”, by fusing with receptors of a typethat do transduce cell growth signals. More specifically, a chimericreceptor that comprises an extracellular region of a ligand-bindingreceptor, and an intracellular region of a type of receptor thattransduces cell growth signals can be used. These chimeric receptorstransduce cell growth signals on binding with a ligand. Receptorssuitable for constructing chimeric receptors by fusion withligand-binding receptors are not especially limited as long as they areof a type that transduces cell growth signals. Receptors that can beused include cytokine receptors and receptors known to those skilled inthe art. G-CSF receptor, mpl, neu, GM-CSF receptor, EPO receptor, c-Kit,FLT-3 and such are specific examples of such receptors. A suitableexample of the above cells that grow ligand (factor)-dependently is aBaF3 ligand (factor)-dependent cell that expresses a chimeric receptorwhose extracellular portion is a ligand receptor chain, and whoseintracellular portion is a G-CSF receptor chain. Other examples of cellsthat can be used in the present invention include NFS60, FDCP-1, FDCP-9CTLL-2, DA-1, KT-3, and such.

As described above, the present invention comprises antibodies, andcells that express receptors which multimerize on binding with thoseantibodies. The present invention also comprises cells whose growth isdependent on ligands (factors) against the receptor.

In the present invention, “antibodies” comprise so-called“immunoglobulins”, as well as natural antibodies, antibody-likemolecules, and antibody fragments. Natural antibodies andimmunoglobulins are generally heterotetramers of about 150,000 Daltonsconsisting of two identical light chains (L chains) and two identicalheavy chains (H chains). Each of the light chains is connected to aheavy chain through a single covalent disulfide bond. However, thenumber of disulfide bonds between the heavy chains varies depending onthe isotype of the immunoglobulin. Both of the heavy and light chainsfurther have intramolecular disulfide bridges at constant distances.Each of the heavy chains has a variable region (V_(H)) at one end andmany constant regions connected thereto. Each of the light chains has avariable region (V_(L)) at one end and a constant region at the otherend. The constant region and the variable region of the light chains areplaced side-by-side to the first constant region and the variable regionof the heavy chain, respectively. Specific amino acid residues areconsidered to form the interface of the variable region of the light andheavy chains (Chothia C. et al., J. Mol. Biol. 186: 651-663, 1985;Novotny J., Haber E., Proc. Natl. Acad. Sci. USA 82: 4592-4596, 1985).In the present invention, a preferable example used as a test antibodyis an immunoglobulin G (IgG).

The above-mentioned phrase “antibody fragment” refers to a part of afull-length antibody and generally indicates an antigen-binding regionor a variable region. For example, antibody fragments include Fab, Fab′,F(ab′)₂ and Fv fragments. Papain digestion of an antibody produces twoidentical antigen-binding fragments called Fab fragments each having anantigen-binding region, and a remaining fragment called “Fc” due toready crystallization. On the other hand, pepsin digestion results in aF(ab′)₂ fragment (which has two antigen-binding sites and can cross bindantigens) and another remaining fragment (called pFc′). Other fragmentsinclude diabody (diabodies), linear antibodies, single-chain antibodies,and multispecific antibodies formed from antibody fragments. In thisspecification, “antibody fragment” of an antibody indicates Fv, F(ab)and F(ab′)₂ fragments, and such.

Herein, an “Fv” fragment is the smallest antibody fragment and containsa complete antigen recognition site and a binding site. This region is adimer (V_(H)-V_(L) dimer) wherein the variable regions of each of theheavy chain and light chain are strongly connected by a noncovalentbond. The three CDRs of each of the variable regions interact with eachother to form an antigen-binding site on the surface of the V_(H)-V_(L)dimer. Six CDRs confer the antigen-binding site of an antibody. However,a variable region (or a half of Fv which contains only three CDRsspecific to an antigen) alone has also the ability to recognize and bindan antigen although its affinity is lower than the affinity of theentire binding site.

Moreover, a Fab fragment (also referred to as F(ab)) further includesthe constant region of the light chain and a constant region (C_(H)1) ofthe heavy chain. An Fab′ fragment differs from the Fab fragment in thatit additionally has several residues derived from the carboxyl end ofthe heavy chain C_(H)1 region which contains one or more cysteines fromthe hinge domain of the antibody. Fab′-SH indicates an Fab′ wherein oneor more cysteine residues of the constant region has a free thiol-group.The F(ab′) fragment is produced by the cleavage of disulfide bondsbetween the cystines in the hinge region of the F(ab′)₂ pepsin digest.Other chemically bound antibody fragments are also known to thoseskilled in art.

In the present invention, diabodies (Db) mean bivalent antibodyfragments constructed by gene fusion (Holliger P. et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO93/11161). Diabodiesare dimers comprising two polypeptide chains, where each polypeptidechain comprises a light chain variable domain (V_(L)) and a heavy chainvariable domain (V_(H)) connected with a linker short enough to preventinteraction of these two domains, for example a linker of about fiveamino acids. The V_(L) and V_(H) domains encoded on the same polypeptidechain will form a dimer because the linker between the V_(L) and V_(H)is too short to form a single chain variable region fragment. Thus, theresult is a diabody that comprises two antigen-binding sites. If theV_(L) and V_(H) domains directed against two different antigens (a andb) are expressed simultaneously as combinations of V_(L)a-V_(H)b andV_(L)b-V_(H)a connected with a linker of about five residues, they canbe secreted as a bispecific diabody.

A single-chain antibody (hereafter also described to as sc Fv) or sFvantibody fragment contains the V_(H) and V_(L) regions of an antibody,and these regions exist on a single polypeptide chain. Generally, an Fvpolypeptide further contains a polypeptide linker between the V_(H) andV_(L) regions, and therefore an scFv can form a structure necessary forantigen binding. See, Pluckthun “The Pharmacology of MonoclonalAntibodies” Vol. 113 (Rosenburg and Moore eds. (Springer Verlag, NewYork) pp. 269-315, 1994) for a review on scFv. In the present invention,linkers are not especially limited as long as they do not inhibitexpression of antibody variable regions linked at their ends.

In addition, a technology using gene engineering to create “humanizedantibodies” is known. In this technology, all but the CDR of monoclonalantibodies from non-human mammals (such as mice, rats, and hamsters) isreplaced with frame structure sequences of variable domains from humanimmunoglobulins (see for example, Jones et al., Nature 321: 522-525(1986); Riechmann et al., Nature 332: 323-329 (1988); Presta, Curr. Op.Struct. Biol. 2: 593-596 (1992)). Humanized antibodies may compriseamino acids that are comprised in neither the CDR introduced into therecipient antibody nor the frame structure sequences. Normally, suchintroduction of amino acid residues is performed to optimize antibodiesfor more precise antigen recognition and binding. The variable domainsin the test antibodies of the present invention also comprise alteredvariable domains, such as humanized domains.

In the present invention, suitable examples of the test antibodies thatare expressed in cells by antibody libraries include IgG, antibodymolecules where scFV is attached at the N-terminal of CH1-hinge-CH2-CH3,scIgG (antibody molecules where scFab is attached at the N-terminal ofhinge-CH2-CH3), scDb, and such. In the present invention, usuallyantibody libraries against each of the different antigens are superinfected into cells; however, when using scDb, a single library can beused as the antibody library.

In a preferable embodiment of the present invention, antibody librariesare introduced into cells. When the antibodies are multi-specificantibodies, antibody libraries against each of the first receptor chainand the second receptor chain are introduced into cells. Retrovirusantibody libraries, for example, can be suitably used as these antibodylibraries. Retroviruses comprise the following features: retroviralinfection efficiency can be controlled to about 10%, so most of theinfected cells can be expected to incorporate one copy of the virus. Inaddition, introduced genes can be incorporated into host chromosomes, sostable expression of these genes over a long period can also beexpected. Other viral vectors that can be produced by antibody librariesinclude RNA viruses and DNA viruses such as adenoviruses,adeno-associated viruses, herpes viruses, vaccinia viruses, pox viruses,sindbis viruses, sendai viruses, SV40 and HIV.

The antibody libraries can be constructed by known methods (see forexample, McCafferty et al., Nature 348: 55-554 (1990); Clackson et al.,Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 582-597(1991), etc). More specifically, antibody libraries can be constructedas follows, but the construction methods are not limited to these.First, mice are immunized with either A-chain or B-chain receptors, andmRNAs are extracted from the splenocytes of these animals. The L-chainand H-chain variable regions are then recovered by RT-PCR using primersthat correspond to mice CDRs. Single chain variable regions (scFv) aresynthesized using assembly PCR to construct antibody libraries.Antigen-binding antibody clones are concentrated by panning, thesesingle chain variable regions obtained from concentrated clones areinserted between a signal sequence for animal cells andCH1-hinge-CH2-CH3, and plasmids into which libraries are incorporatedfor generating retroviruses are constructed. Alternatively, scFab issynthesized, and libraries inserted between a signal sequence andhinge-CH2-CH3 are constructed. scDb libraries can also be made.

The antibody libraries of the present invention can also be constructedusing plasmid expression vectors. Examples of expression vectors whenthe above cells are animal cells include pME18S (Med. Immunol. 20: 27-32(1990)), pEF-BOS (Nucleic Acids Res. 18: 5322 (1990)), pCDM8 (Nature329: 840-842 (1987)), pRSVneo, pSV2-neo, pcDNAI/Amp (Invitrogen),pcDNAI, pAMoERC3Sc, pCDM8 (Nature 329: 840 (1987)), pAGE107(Cytotechnology 3: 133 (1990)), pREP4 (Invitrogen), pAGE103 (J. Biochem.101: 1307 (1987)), pAMoA, pAS3-3, pCAGGS (Gene 108: 193-200 (1991)),pBK-CMV, pcDNA3.1 (Invitrogen), and pZeoSV (Stratagene). Expressionpromoters may be cytomegalovirus IE gene promoter and enhancer, SV40early promoter, a retrovirus LTR such as that from RSV, HIV, and MMLV,and a gene promoter from animal cells such as metallothionein, β-actin,elongation factor-1, HSP genes, and such. Alternatively, viral vectorsmay be used as above. Viral vectors may be DNA viruses and RNA virusessuch as retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, Simbu viruses, Sendai viruses,SV40, and HIV.

The methods for introducing plasmid expression vectors may varydepending on the type of cell and vector. Any method can be used as longas expression vector DNA can be introduced into cells. For example, themethods include electroporation (Cytotechnology 3: 133 (1990)), calciumphosphate (JP-A Hei 2-227075), lipofection (Proc. Natl. Acad. Sci. USA84: 7413 (1987); Virology 52: 456 (1973)), co-precipitation with calciumphosphate, DEAE-dextran, direct injection of DNA using microcapillaries,and such.

Furthermore, where the receptors are heteroreceptors (where theantibodies are multi-specific antibodies), expression of the testantibodies of the present invention is usually possible by introducing,into one cell, expression libraries (vectors) for each of the two typesof light chains and two types of heavy chains. No compatibility isusually present for the light and heavy chain combinations. For fourchains, there are ten types of combinations. The agonistic antibodiesfinally screened by the methods of the present invention are preferablywhole antibodies that are stable in the blood. Thus, it is preferablethat the test antibodies used in the screening methods of the presentinvention are also full length. When, as above, libraries are expressedin cells as is, ten different types of antibodies are secreted. Due tothe potential of neutralizing effects by other antibody combinations, itis preferable to express test antibodies that comprise the desiredcombination of heavy and light chains. To get the desired combination,for example, a common light chain is preferably used. Alternatively, toavoid homogenous combinations of heavy chains, it is also possible toencourage heterogeneous combination using amino acid substitution tointroduce “knobs-into-holes” (Protein Engineering Vol. 9, 617-621, 1996;WO98/50431) at heavy chain CH3 regions, thus blocking the formation ofheavy chain homodimers. Thus in the present invention, test antibodyCH3s are preferably characterized by the introduction of“knobs-into-holes” by amino acid substitution. Appropriate amino acidsubstitutions are carried out in the CL and CH1, and by defining thecombinations of light and heavy chains, the production efficiency ofhetero-antibodies can be raised. Furthermore, combinations of light andheavy chains can be defined using scFv single-chain variable regionsthat link the light and heavy chain variable regions with linkers. Testantibodies can also be expressed in the form of scFv-CH1-hinge-CH2-CH3.Therefore, in one embodiment of the present invention, the testantibodies are characterized by the linking of their light and heavychain variable regions by linkers. These multi-specific test antibodieswith variable regions linked by linkers can be produced, for example, asbelow. However, production is not particularly limited to these methods.

-   (1) A single chain Fv against the first receptor chain is produced.    Next, by linking that single chain Fv with CH1-hinge-CH2-CH3, a    single chain antibody against the first receptor chain is produced.    Test antibodies comprising that single chain antibody are then    produced.-   (2) A single chain Fab against the first receptor chain is produced.    Next, by linking that single chain Fab with Fc, a single chain    antibody against the first receptor chain is produced. Test    antibodies comprising that single chain antibody are then produced.

In addition, the present invention's screening methods formulti-specific agonistic antibodies can be provided by usingmulti-specific antibodies, produced by known manufacturing methods, astest antibodies.

The “contact” of test antibodies and receptors in the above step (a) inthe present invention can refer to, for example, the “contact” ofreceptors expressed in the cells with test antibodies expressed in andsecreted out of the cells as mentioned above. However, “contact” is notparticularly limited to this form of contact.

In the above-mentioned screening methods of the present invention,whether or not a test antibody comprises agonistic activity is thendetermined (step b), and antibodies comprising agonistic activity areselected (step c).

In the above-mentioned steps in a preferable embodiment of the presentinvention, whether or not test antibodies comprise agonistic activity isjudged by using the presence or absence of the above-mentionedautonomous autocrine cell replication as an index. Where there isautonomous autocrine cell replication, antibodies expressed by thosecells are selected as agonistic antibodies.

Agonistic antibodies selected in the screening methods of the presentinvention are usually recovered from cells with autonomous autocrinereplication, and cloned accordingly. These cells are used to recover theCDR genes of the above-mentioned multi-specific agonistic antibodies byPCR, and these recovered genes can then be used for antibody production.One skilled in the art can use routine procedures to ordinarily carryout the production of antibodies based on the CDR genes.

Where the receptor is a heteroreceptor, the screening methods formulti-specific antibodies of the present invention can be carried outspecifically as below. However, this method is one embodiment of thepresent invention, and the methods of the present invention are notespecially limited to this method.

Antibody phage libraries are produced from animals immunized with theextracellular region of receptor chains that use antibodies as ligands(e.g. the A chain and B chain). Anti-receptor chain antibodies areselected by panning, and then transformed to retroviral vectorlibraries.

Next, a cell line that proliferates ligand-dependently is prepared. Ananti-A-chain antibody library is introduced into these cells byinfection, and infected cells are selected using a drug-resistant geneincorporated into the vector. Selected cells are then cultured andsubsequently, super-infected with the anti-B-chain antibody library. Inthis way, a library of cells expressing bispecific antibodies of everyanti-A-chain antibody and anti-B-chain antibody combination can beconstructed. Of these, only clones that secrete appropriately combinedbispecific antibodies showing agonistic activity against a targetreceptor can autonomously replicate upon autocrine stimulation. In thisway, the engineered antibody genes can be recovered by PCR from thechromosomes of selected BaF3 clones.

Methods of screening for antibodies that comprise agonistic activity byusing autonomous autocrine growth were unknown until now, and hence werenewly discovered by the present inventors. The present inventionprovides agonistic antibody screening methods that use autonomousautocrine replication due to common antibodies, including multi-specificantibodies.

The above-mentioned screening methods first provide cells that express atest antibody and a receptor that multimerizes, where the growth ofthose cells depends on a ligand (factor) of that receptor (step A).Next, where the cells undergo autonomous autocrine replication, the testantibodies are judged to comprise agonistic activity (step B).Antibodies that comprise agonistic activity are then selected (step C).

The above-mentioned methods are not particularly limited to thescreening of multi-specific agonistic antibodies, and screening foragonistic antibodies that do not show multi-specificity is alsocomprised in the above-mentioned methods.

It is commonly known that light chains do not have much effect onantibody specificity. Thus, even when antibodies are multi-specific, theabove-mentioned methods can be carried out without introducing two typesof heavy chains and two types of light chains into cells. Theabove-mentioned methods can also be carried out by introducing two kindsof heavy chain libraries into cells after pre-expressing one kind oflight chain. Thus, a preferable embodiment of the present invention, inaddition to steps (A) to (C), comprises the step of introducing genesthat code for a test antibody's heavy chain into the cells of step (A),which have been introduced with a gene coding for the receptor, and agene coding for the test antibody's light chain.

In addition, the present invention provides methods for producingagonistic antibodies that use the methods of screening for agonisticantibodies of the present invention.

In the above methods, agonistic antibodies are first screened by themethods of screening for agonistic antibodies of the present invention,and genes that code for the agonistic antibodies selected by thisscreening are introduced into host cells. Multi-specific agonisticantibodies are then recovered from those host cells or their culturesupernatant.

The antibodies of the present invention, obtained as above, can beisolated using signal sequences from inside cells, or from the culturemedium if secreted into the extracellular space. They can then bepurified as substantially pure polypeptides. Separation and purificationof polypeptides can be performed by appropriately selecting or combiningmethods as necessary. Separation methods can be selected from thosegenerally used, such as chromatography, filtration, ultrafiltration,salting out, solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricpoint focusing, dialysis, and recrystallization. Chromatography includesaffinity chromatography, ion exchange chromatography, hydrophobicchromatography, gel filtration, reverse phase chromatography, absorptionchromatography, and the like (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual, Daniel R. Marshak et al.eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: ALaboratory Course Manual, Harlow and David Lane eds., Cold Spring HarborLaboratory Press (1988)). Such chromatographies may be performed usingliquid chromatographies such as HPLC, FPLC, and the like. In addition,since the antibodies of the present invention bind antigens, they may bepurified by making use of this ability. Agonistic antibodies isolatedand purified in this way are themselves included in the presentinvention.

The test antibodies in a preferable embodiment of the present inventionhave their heavy and light chain variable regions linked by linkers, andare introduced with “knobs-into-holes” by amino acid substitution atCH3. Thus, the antibodies provided by the present invention arepreferably agonistic antibodies that are characterized by having theirheavy and light chain antibody variable regions linked by linkers, andthat are introduced with “knobs-into-holes” by amino acid substitutionat CH3 of the antibodies.

In the screening methods of the present invention, the agonist activitythat can be used as a detection index is preferably proliferationactivity. However, it is also possible to use, for example, changes inthe amount and/or type of substance produced (secretary proteins,surface antigens, intracellular proteins, mRNA, etc.), anchoragedependency, cytokine-dependant responsiveness, hormone dependency, drugresistance, cell motility, cell migration activity, pulsatility, changesin intracellular substance, protein phosphorylation, etc.

Agonistic antibodies that can be acquired by the screening methods ofthe present invention can be used as pharmaceutical agents forimmunotherapy or prevention, in the same way as traditionally knownmulti-specific antibodies. IL-10, 12, 24, 4, 7, 9, 13, TSLP, IFNα, β andsuch are known as ligands for immune system receptors made up ofheterodimers. NGF, GDNF, NT-3, 4 and 5 are known as ligands for nervoussystem receptors. Antibodies acquired by the methods of the presentinvention can be, for example, antibodies that comprise anabove-mentioned ligand-like function. The antibodies acquired by themethods of the present invention are expected to become pharmaceuticalsfor therapy of immune or nervous system illnesses and such.

Pharmaceutical compositions used for such therapeutic or preventivepurposes, which comprise antibodies of the present invention as activeingredients, may be formulated by mixing with suitable pharmaceuticallyacceptable carriers and solvents, if necessary, that are inactiveagainst the antibodies. For example, sterilized water, saline,stabilizers, vehicles, antioxidants (e.g. ascorbic acid), buffers (e.g.phosphate, citrate, other organic acids), preservatives, detergents(e.g. PEG, Tween), chelating agents (e.g. EDTA), and binders may beused. Alternatively, they may comprise other low molecular weightpolypeptides, proteins such as serum albumin, gelatin andimmunoglobulins, amino acids such as glycine, glutamine, asparagine,arginine, and lysine, carbohydrates and sugars such as polysaccharidesand monosaccharides, and sugar alcohol such as mannitol and sorbitol.When prepared as an aqueous solution for injection, saline and isotonicsolutions comprising glucose and other adjunctive agents such asD-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used. Inaddition, an appropriate solubilizing agent such as alcohol (e.g.ethanol), polyalcohol (e.g. propylene glycol, PEG), and non-ionicdetergents (e.g. polysorbate 80, HCO-50) may be used in combination.

If necessary, antibodies of the present invention may be encapsulated inmicrocapsules (microcapsules made of hydroxycellulose, gelatin,polymethylmethacrylate, and the like), and made into components ofcolloidal drug delivery systems (liposome, albumin microsphere,microemulsion, nano-particles, and nano-capsules) (refer to, forexample, “Remington's Pharmaceutical Science 16th edition”, Oslo Ed.(1980)). Moreover, methods for making sustained-transduce drugs areknown, and these can be applied for the antibodies of the presentinvention (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981);Langer, Chem. Tech. 12: 98-105 (1982); U.S. Pat. No. 3,773,919; EPPatent Application No. 58,481; Sidman et al., Biopolymers 22: 547-556(1983); EP: 133,988).

Administration to patients may be preferably performed by injections orintravenous drips; for example, in addition to intra-arterialinjections, intravenous injections, and subcutaneous injections, methodsknown to one skilled in the art may be used, such as administratingintranasally, intrabronchially, intramuscularly, percutaneously, ororally. Doses may vary depending on various factors, including patientbody weight and age, type of disease, symptoms, and administrationmethod, but those skilled in the art are able to appropriately selectsuitable doses.

In addition, genes encoding antibodies of the present invention may beused for gene therapy by cloning into vectors for such use. Such vectorscan be administered by direct injection using naked plasmids, and alsoby packaging in liposomes, producing as a variety of viral vectors suchas retrovirus vectors, adenovirus vectors, vaccinia virus vectors,poxvirus vectors, adenoassociated virus vectors, and HVJ vector (Adolph,“Virus Genome Methods”, CRC Press, Florida (1996)), or coating ontocarrier beads such as colloidal gold particles (e.g. WO93/17706).However, any method can be used for administration as long as theantibodies are expressed in vivo and exercise their function.Preferably, a sufficient dose may be administered by a suitableparenteral route (such as injecting intravenously, intraventricularly,subcutaneously, percutaneously, or into adipose tissues or mammaryglands, inhalation, intramuscular injection, infusion, gas-inducedparticle bombardment (using electron gun and such), or through themucosa (for example, by nose drops). Alternatively, genes encodingantibodies of the present invention may be administered into, forexample, blood cells and bone marrow cells ex vivo using liposometransfection, particle bombardment (U.S. Pat. No. 4,945,050), or viralinfection, and the cells may be reintroduced into animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Conditioned medium (CM)-dependent growth of anautonomously replicating cell line. 200 μL of various concentrations ofculture supernatants of MPL-expressing BaF3 cells that acquiredautonomous replication by viral infection, was added to each wellcontaining 10,000 washed MPL-expressing BaF3 cells (cells expressingchimeric receptors of the MPL extracellular region, and GCSF receptortransmembrane/intracellular region: TPG). After three days, 20 μL of SFreagent (Nacarai Tesque) for measuring the viable cell number was added.After two and a half hours, absorbance was measured at 450 nm, and thenumber of viable cells was investigated. MPL-expressing BaF3 cells(HL(TPG) and LH(TPG)) maintained cell growth in a culture supernatantconcentration-dependant manner. On the other hand, parent cell linesBaF3 (HL(BaF3) and LH(BaF3)), which do not express receptors, did notgrow, since the antibodies do not act on these cells. The x-axis showsConditioned medium (CM) concentration (%). The z-axis shows absorbanceat 450-655 nm.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described withreference to Examples, but it is not to be construed as being limitedthereto.

EXAMPLE 1 Autocrine Growth by Diabodies

As a model of agonistic antibodies, the anti-mpl-monoclonal antibody12E10 (WO99/10494) was used. An EcoRI-NotI fragment encoding a diabodywas excised from pCOSsc12E10 (WO01/79494, WO02/33072) encoding theantibody 12E10 variable region. This fragment was inserted between theEcoRI and NotI of viral vector plasmid pMX. This plasmid pMXsc12E10 wastransfected into Pt—B packaging cells using FuGene6 (Roche). Pt—B cellswere seeded in 6 cm dishes containing Dulbecco's Modified Eagle Medium(DMEM) with 10% fetal calf serum (FCS). The next day a mixture ofFuGene6 and plasmid pMXsc12E10 was added to the culture. A day afterthat, the culture medium was replaced, and the culture supernatant wascollected after 24 hours. 10 μg/mL of Polybrene (Hexadimethrine Bromide,Sigma) was added to culture supernatant containing the recombinantvirus, target cells were suspended in this culture supernatant andinfected. Human MPL cDNA was introduced into ligand (factor)-dependentcell line BaF3. Cell line MPL/BaF3 (cells expressing chimeric receptorsof the MPL extracellular region, and GCSF receptortransmembrane/intracellular region), which can grow due to the additionof MPL ligand (thrombopoietin), was infected by adding a viral solutionwith mouse interleukin-3 (IL-3). The next day the cells were washed withPBS. Culture was continued in IL-3-free RPMI with 10% FCS, andautonomously replicating cells were obtained. Where the culturesupernatant of these cells was collected and added to different MPL/BaF3for incubation, a cell growth dependant on the concentration of theadded culture supernatant was observed. From this it was clear that theautonomous replication of virus-infected cells was due to the autocrinestimulation of diabodies secreted into the culture medium by thesecells.

EXAMPLE 2 Autocrine Growth by scFv-CH1-Fc

PCR was carried out using pCOSsc12B5 as a template, and primers EcoRI-HL(5′-GGAATTCGCCGCCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCT-3′: SEQ ID NO: 1) andHL-SfiI(5′-GCATGCATGGCCCCCGAGGCCACTCACCTTTGATCTCCAGCTTGGTCCCTCCGCCGAA-3′: SEQID NO: 2). An scFv (H-L) gene comprising an EcoRI site at its 5′ and asplice donor sequence and SfiI site at its 3′ was obtained. In addition,to obtain a gene connected in the light-heavy (L-H) chain order, PCR wascarried out using pCOSsc12B5 as a template, and a primer combination of5Hs(5′-GGCGGCGGCGGCTCCGGTGGTGGTGGATCCCAGGTGCAGCTGGTGCAGTCTGG-3′: SEQ IDNO: 3) and 3Ha-SfiI(5′-GCATGCATGGCCCCCGAGGCCACTCACCTGAAGAGACGGTGACCATTGTCCCTT-3′: SEQ IDNO: 4); or5Ls(5′-AGTCAGTCGGCCCAGCCGGCCATGGCGGACTACAAAGACATCCAGATGACCCAGTCTCCT-3′:SEQ ID NO: 5) and 3La(5′-GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCTTTGATCTCCAGCTTGGTCCCTCCGCCGAAA-3′: SEQ ID NO: 6). By again carrying out PCR on bothobtained amplification products using primers 5Hs and 3La, theamplification products were assembled. The produced LH comprised an SfiIsite on both ends, and a splice donor site at the 3′ side.

PCR was carried out using plasmid HEF-1.24H-gγ1 (WO99/18212) as atemplate, which comprises a human IgG1 constant region gene. The intronbefore CH1 and CH1; hinge (primers EcoSfiI(5′-TCGAATTCGGCCTCGGGGGCCAGCTTTCTGGGGCAGGCCAGGCCTGACCTTGGCTTT-3′: SEQ IDNO: 7), and HigeCH1a(5′-CACGGTGGGCATGTGTGAGTTTTGTCACAAGATTTGGGCTCAACTTTCTTGTCCACCTTG-3′: SEQID NO: 8)); CH2 (primers HigeCh2s(5′-CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT-3′: SEQID NO: 9), and Ch3Ch2a(5′-ACACCTGTGGTTCTCGGGGCTGCCCTTTGGCTTTGGAGATGGTTTTCTCGATG-3′: SEQ ID NO:10)); CH3 (primers Ch2Ch3sW (5′-GGCAGCCCCGAGAACCACAGGTGTACACCC-3′: SEQID NO: 11), and StopNotI(5′-TAGCGGCCGCTCATTTACCCGGAGACAGGGAGAGGCTCTT-3′: SEQ ID NO: 12)) wereamplified. By continuing with this assembly PCR, an IgG1 constant regiongene comprising an EcoRI site, an SfiI site, and an intron at its 5′,and a NotI site at its 3′ was constructed.

PCR was carried out using mouse interleukin (IL)-3 cDNA as a template,and primers IL3sEcoA (5′-CGGAATTCGGCCGGCTGGGCCAGCATCAGGAGCAGGAGCAGC-3′:SEQ ID NO: 13), and BamIL3ss(5′-GCGGATCCGCCGCCACCATGGTTCTTGCCAGCTCTAC-3′: SEQ ID NO: 14), and an IL3signal sequence (ss) gene comprising a BamHI site at its 5′, and an SfiIsite and EcoRI site at the 3′ was obtained.

The synthesized IgG1 constant region gene was inserted in the EcoRI-NotIsite of a retroviral vector pMX, and pMX-CHwild was constructed.pMX-HL-CHwild was obtained by incorporating the scFv (H-L) gene into theEcoRI-SfiI site of the pMX-CHwild plasmid. When the virus derived fromthis plasmid infects cells, molecules used as antibodies, which haveCH1-hinge-CH2-CH3 linked with the scFV (H-L) C-terminal of antibody12B5, can be expected to be secreted. Then, IL3ss was incorporated intothe BamH1-EcoRI site of pMX-CHwild, and scFv (L-H) was furtherincorporated into the SfiI site to construct pMX-IL3ss-LH-CHwild.ScFv-CH1-hinge-CH2-CH3 (L-H) can be expected to be secreted by thisplasmid-derived virus. The above plasmids were respectively transfectedinto Pt-E cells, as above. Recombinant viruses were obtained and used toinfect MPL/BaF3 cells. The next day, IL-3 was removed by washing, andculture was continued. As a result, cells that can replicateautonomously were obtained. The culture supernatant of these cells wascollected, and on culturing with different MPL/BaF3s, cell growthdependant on the concentration of culture supernatant added was observed(FIG. 1, TPG). On the other hand, parent line BaF3 did not express thereceptors, and thus did not proliferate as antibodies did not act onthem (FIG. 1, BaF3). From this, the autonomous replication ofvirus-infected cells was revealed to be due to the autocrine stimulationof scFv-CH1-hinge-CH2-CH3 secreted into the medium by those cells.

INDUSTRIAL APPLICABILITY

The present invention provides novel methods that can effectively screenfor agonistic antibodies. In a preferable embodiment of the presentinvention, screening is made easily possible by using antibodies aslibraries, without the need for complicated procedures. In addition, ina preferable embodiment of the present invention, autonomous autocrinereplication is used as an index for screening. Since only clones thatsecrete agonistic antibodies themselves are selected as cells that havereproduced via receptor signals, multiple samples (antibody libraries)can be treated simultaneously, and extremely effectively.

1. A method of screening for agonistic antibodies, the methodcomprising: (a) providing a cell that expresses both a multimer-formingreceptor and a test antibody, wherein the cell in the absence of theantibody requires a ligand of the receptor for growth; (b) culturing thecell in the absence of the ligand; and (c) selecting the test antibodyas an agonist of the receptor if the cell grows in the absence of theligand.
 2. The method of claim 1, flirt her comprising the steps of (i)providing a cell comprising a nucleic acid encoding the light chain ofthe antibody and a nucleic acid encoding the receptor; and (ii)introducing into the cell a nucleic acid that encodes the heavy chain ofthe test antibody, thereby producing the cell of step (a).
 3. The methodof claim 1, wherein the receptor is a chimeric receptor that functionsto transduce a cell growth signal.
 4. The method of claim 1, wherein thereceptor is a dimer-forming receptor.
 5. The method of claim 4, whereinthe dimer-forming receptor is a homo-dimer-forming receptor.
 6. Themethod of claim 4, wherein the dimer-forming receptor is ahetero-dimer-forming receptor.
 7. The method of claim 1, wherein thereceptor is a G-CSF receptor.
 8. The method of claim 1, furthercomprising a step of producing a plurality of cells expressing a libraryof diverse antibodies, the cell of step (a) being a member of theplurality of cells.
 9. The method of claim 8, wherein the library ofdiverse antibodies is encoded by a retroviral antibody libraryintroduced into the plurality of cells.
 10. The method of claim 1,wherein the test antibody is a multi-specific antibody.
 11. The methodof claim 10, wherein the test antibody comprises heavy and light chainvariable regions connected via a linker.
 12. The method of claim 11,further comprising expressing the test antibody by a method thatcomprises: (i) producing a first DNA encoding a single chain Fv thatbinds to the receptor; (ii) producing a second DNA encoding a singlechain antibody comprising the single chain Fv of step (i) linked to aCH1-hinge-CH2-CH3; and (iii) expressing a multi-specific antibody thatcomprises the single chain antibody of step (ii).
 13. The method ofclaim 11, further comprising expressing the test antibody by a methodthat comprises: (i) producing a first DNA encoding a single chain Fabthat binds to the receptor; (ii) producing a second DNA encoding asingle chain antibody comprising the single chain Fab of step (i) linkedto an Fc; and (iii) expressing a multi-specific antibody that comprisesthe single chain antibody of step (ii).
 14. A method of screening foragonistic antibodies, the method comprising: providing an antibodyexpression library of cells, the cells each expressing both a member ofa set of diverse antibodies and a multimer-forming receptor, wherein thecells in the absence of the antibodies require a ligand of the receptorfor cell growth; culturing the library of cells in the absence of theligand; selecting a cell that grows in the absence of the ligand; andidentifying the antibody expressed by the selected cell as being anagonist of the receptor.
 15. The method of claim 14, wherein theantibody expression library comprises a retroviral antibody library.